The present invention relates generally to a method for the preparation of uniformly sized populations of lipid vesicles, liposomes, which may be used to encapsulate aqueous materials.
Liposomes are spherical shells of amphipathic molecules which isolate an interior aqueous space from the bulk exterior aqueous environment and are characterized by their lipid composition, the most commonly used lipid component being phospholipid. Because liposomes can be made to contain hydrophobic molecules within their membrane, or hydrophilic markers within their internal aqueous space, or both, liposomes can be used as potential vehicles for the delivery of drugs in vivo and as the basis for immunoassay systems in vitro such as the Liposome Immuno-Lytic Assay (LILA) which involves the antibody-triggered complement-mediated lysis of liposomes. For use in such complement-mediated immunoassays, liposomes should be homogeneous in size distribution, be small enough to remain in suspension, and be large enough to entrap sufficient marker, such as fluorophore or enzyme; to provide a high signal.
Many methods have been described in the literature for making a wide variety of both multilamellar and unilamellar liposomes. See, Szoka, et al., Annu. Rev. Biophys. Biogen., 9:467-508 (1980); Deamer and Uster, "Liposomes" Ch. 1, Liposome Preparation: Methods and Mechanisms; Marc J. Ostro (Ed.) M. Dekker, Inc., pp. 27-51 New York, NY (1983); and Szoka, et al., Proc. Nat'l. Acad. Sci. U.S.A., 75:4194-4198 (1978).
Multilamellar vesicles (MLV) are prepared by depositing lipids from organic solvents in a thin film on the wall of a round-bottom flask by rotary evaporation under reduced pressure followed by hydration with aqueous buffers and agitation. Bangham, A., et al., J. Mol. Biol., 13:238-252 (1965). The hydration time, method of resuspension of lipids, and thickness of the lipid film can result in markedly different preparations of MLVs, despite a constant lipid concentration and composition, and volume of suspending aqueous phase. The size ranges obtainable by this method vary from 0.4 to 50 microns (400 to 50,000 nanometers). Papahadjopoulos and Miller, Biochem. et Biophy. Acta., 135:624 (1967). Szoka, F., et al., Proc. Nat'l. Acad. Sci. U.S.A., 75:4194-4198 (1978).
Mezei, et al., U.S. Pat. No. 4,485,054, disclose a procedure for producing large multilamellar lipid vesicles (MLV) which may be used to encapsulate biologically active materials, particularly lipophilic substances. The lipid film forming step is conducted in a vessel partially filled with inert, solid, contact masses, in particular, spherical contact masses having a diameter of 1.0 mm to 100 mm (1,000 to 100,000 microns) and results in liposomes reported to range in diameter from 5 to 10 microns (5,000 to 10,000 nanometers).
MLVs are relatively easy to prepare, can be made from a wide variety of lipid compositions, and are efficiently lysed by complement. Because they have many lamellae, they are stable against leakage and therefore have good shelf life. However, the use of MLVs can be disadvantageous because MLV preparations typically are heterogeneous in size distribution, in shape, and in entrapped volume. For in vivo applications such as drug delivery, the largest MLVs are most readily filtered from the blood stream and consequently most rapidly sequestered in the lungs and reticulo-endothelial system, thereby limiting the circulating half-life of liposome-entrapped drugs and hindering efforts towards tissue specific drug targetting. Scherphof, et al., Biochem. Soc. Trans., 15:345-348 (1987); and Juliano and Stamp, Bioch. Biophys. Res. Comm., 63:651-658 (1975).
When used in complement-mediated assays, the largest MLVs tend to settle, thereby changing the distribution of MLVs in solution and causing a shift in the immunoassay standard curve. Further, liposome lysis varies as a function of both the size and the type of liposome used in the immunoassay. Richards, et al., Biochem. et Biophys. Acta., 855:223-230 (1986). Liposome interaction with plasma also varies as a function of the size and type of liposome. Scherphof, et al., Liposome Technology, Volume III, pp. 205-224 Edited by G. Gregnodia, CRC Press Boca Raton, Florida (1984). These results indicate that liposome size is an important parameter which needs to be controlled in order to obtain reproducible complement mediated lysis.
Although the heterogeneous size problem can be overcome by sizing by passage through filters, the filters tend to clog when high concentrations of lipid and large volumes of liposomes are filtered. Clogging is also a function of the lipid composition, e.g., the charge of the lipids used to prepare the liposomes, the interaction with the filter material, and the tendency of the liposomes to aggregate during and after filtration. Frequent changes of filters is both difficult and inconvenient. Generally, filtration is more suitable for research scale preparations rather than production scale preparations. Furthermore, a high lipid concentration is necessary for efficient drug encapsulation if liposomes are to be used for drug delivery.
Despite substantial research and development in methods for producing liposome (especially MLV) preparations for immunoassay and drug delivery use, there continues to exist a need in the art for new, rapid and simple preparatives techniques which are: (1) applicable to liposome formation using widely varying lipid components and concentrations of the same; (2) applicable liposome entrapment of aqueous compositions of widely varying chemical compositions; (3) capable of providing storage stable, substantially homogeneous populations of small sized lipsomes without resort to cumbersome "screening" processes; and (4) susceptible to use in both small and large scale liposome production procedures.